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Biomedical subjects

D H Ingbar

Publications and source records attributed to D H Ingbar.

At least 37 records · Page 2Linked to original sources
PubMed1998

Adrenergic stimulation of Na+ transport across alveolar epithelial cells involves activation of apical Cl- channels.

Alveolar epithelial cells were isolated from adult Sprague-Dawley rats and grown to confluence on membrane filters. Most of the basal short-circuit current (Isc; 60%) was inhibited by amiloride (IC50 0. 96 microM) or benzamil (IC50 0.5 microM). Basolateral addition of terbutaline (2 microM) produced a rapid decrease in Isc, followed by a slow recovery back to its initial amplitude. When Cl- was replaced with methanesulfonic acid, the basal Isc was reduced and the response to terbutaline was inhibited. In permeabilized monolayer experiments, both terbutaline and amiloride produced sustained decreases in current. The current-voltage relationship of the terbutaline-sensitive current had a reversal potential of -28 mV. Increasing Cl- concentration in the basolateral solution shifted the reversal potential to more depolarized voltages. These results were consistent with the existence of a terbutaline-activated Cl- conductance in the apical membrane. Terbutaline did not increase the amiloride-sensitive Na+ conductance. We conclude that beta-adrenergic stimulation of adult alveolar epithelial cells results in an increase in apical Cl- permeability and that amiloride-sensitive Na+ channels are not directly affected by this stimulation.

Adrenergic beta-Agonists↗
PubMed1998

Perfusion technique determines alveolar fluid resorption rate in the isolated perfused rat lung.

The isolated perfused lung (IPL) preparation is a well-established model for the study of alveolar epithelial sodium transport. We noted that preparations of normal fluid-filled rat lungs with recirculated perfusate reproducibly lost weight, whereas preparations in which the perfusate was discarded after a single pass through the lungs had a variable and lesser weight change. To confirm this, we performed IPL experiments by using male Sprague-Dawley specific-pathogen-free rats (175-225 g). In 10 IPLs, perfusate initially was discarded after passing through the lungs and then was recirculated continuously. During the single-pass period, the rate of weight change was +0.7 +/- 2.0 mg/min compared with -9.0 +/- 1.3 mg/min for the recirculating period. Adenosine 3',5'-cyclic monophosphate (cAMP) accumulated during recirculation. The weight loss induced by recirculation was reproduced by perfusion with 8-bromoadenosine 3',5'-cyclic monophosphate or terbutaline in single-pass fashion and blocked when the kinase inhibitor H-8 or phosphodiesterase was present in the recirculating perfusate. In summary, perfusate recirculation in the IPL stimulates fluid resorption at least partially via cAMP. This should be factored into the design and interpretation of IPL experiments.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

The effects of hyperoxic injury and antioxidant vitamins on death and proliferation of human small airway epithelial cells.

Previously it was reported that hyperoxia induced death of the human lung adenocarcinoma cell line (A549 cells) by necrosis, not by apoptosis. This study examined proliferation and death of untransformed human small airway epithelial (SAE) cells in normoxia or hyperoxia in comparison with A549 cells. We tested the hypothesis that SAE cells respond differently to hyperoxic injury than do A549 cells. We measured total cell number and viability, thymidine incorporation (SAE cells only), lactate dehydrogenase (LDH) release, and apoptotic changes as markers for cell proliferation and death. Protective effects of antioxidant vitamins also were examined in SAE cells. In normoxia, subconfluent SAE cells had less apoptosis and fewer detached cells, but higher thymidine incorporation than did near-confluent cells. Hyperoxia suppressed thymidine incorporation and augmented apoptosis in both subconfluent and near-confluent SAE cells. Hyperoxia decreased the total cell number only in subconfluence, whereas SAE cell viability declined with hyperoxia in near confluence, but not in subconfluence. For SAE cells, necrosis assessed by LDH release was minimal in all conditions and was not augmented by hyperoxia in SAE cells. In contrast, normoxic A549 cells proliferated more rapidly than did SAE cells with a large number of cells detached during the culture. A549 cells underwent necrotic cell death under confluent or in hyperoxic conditions, but had much less apoptotic cell death. In SAE cells, vitamin E partially prevented the decline of thymidine incorporation with hyperoxia in subconfluence and protected against apoptotic changes with hyperoxia in both subconfluent and near-confluent conditions. Vitamin C prevented apoptosis with hyperoxia only in near-confluent SAE cells. Thus, SAE cells maintained balanced apoptosis and cell proliferation that were altered by cell density and hyperoxia and demonstrated very little necrosis with hyperoxia. Although A549 cells underwent cell death mainly by necrosis, they also were influenced by cell density and hyperoxia. Cell density also determined specific antioxidant vitamin protection in SAE cells.

Antioxidants↗
PubMed1998

Oxidant effects on epithelial Na,K-ATPase gene expression and promoter function.

The lung epithelium resorbs alveolar fluid through combined action of sodium channels and the sodium pump, Na,K-ATPase. The lung often is exposed to hyperoxia in disease states and hyperoxia generates a mixture of reactive oxygen species. In vivo and in vitro exposure of rat lung and alveolar type II cells, respectively, increases gene expression of both alpha-1 and beta-1 subunits of the sodium pump. In contrast to the primary type II cells, several type II cell lines did not increase sodium pump gene expression with hyperoxia, but the renal tubular epithelial MDCK cell line did. Using promoter-receptor constructs transfected into MDCK cells, hyperoxia did not markedly increase transcription of the alpha-1 subunit but doubled transcription of the beta-1 subunit gene. Using 5'-deletion constructs, the region required for the beta-1 increase was localized to a 40-base pair region from -44/-84. The hyperoxic responsiveness of this region was confirmed using constructs with one or two copies of this region placed in minimal promoter-luciferase reporters. This 5' promoter region contains a consensus binding sequence for SP-1, a basal transcription factor but not for binding of other known transcription factors. Thus, hyperoxia induces Na,K-ATPase beta-1 promoter transcription, likely acting through a novel mechanism.

Animals↗
PubMed1997

The critical early proinflammatory events associated with idiopathic pneumonia syndrome in irradiated murine allogeneic recipients are due to donor T cell infusion and potentiated by cyclophosphamide.

We have hypothesized that lung damage occurring in the peri-bone marrow transplant (BMT) period is critical for the subsequent generation of idiopathic pneumonia syndrome (IPS), a major complication following human BMT. The proinflammatory events induced by a common pre-BMT conditioning regimen, cyclophosphamide (Cytoxan(R)) (Cy) and total body irradiation, were analyzed in a murine BMT model. Electron microscopy indicated that Cy exacerbated irradiation-induced epithelial cell injury as early as day 3 after BMT. Allogenicity was an important contributing factor to lung injury as measured by lung wet and dry weights and decreased specific lung compliance. The most significant pulmonary dysfunction was seen in mice receiving both allogeneic T cells and Cy conditioning. IPS was associated with an influx of T cells, macrophages, and neutrophils early post-BMT. Hydroxyproline levels were not increased, indicating that the injury was not fibrotic early post-BMT. As early as 2 h after chemoradiation, host macrophages increased in number in the lung parenchyma. Continued increases in macrophages occurred if splenic T cells were administered with the donor graft. The expression of costimulatory B7 molecules correlated with macrophage numbers. Frequencies of cells expressing mRNA for the inflammatory proteins TNF-alpha, IL-1beta, and TGFbeta were increased. Cy accelerated the upregulation of TGFbeta and increase in host macrophages. The exacerbation of macrophage activation and severity of IPS was dependent on allogeneic T cells, implicating immune-mediated mechanisms as critical to the outcome of IPS. This demonstration of early injury after BMT indicates the need for very early therapeutic intervention before lung damage becomes profound and irreversible.

Animals↗
PubMed1997

Cell density and antioxidant vitamins determine the effects of hyperoxia on proliferation and death of MDCK epithelial cells.

Epithelial cells are prone to oxidant injury, which could change epithelial cell homeostasis and lead to degenerative diseases. We examined the effects of hyperoxia on death and proliferation off Madin-Darby canine kidney (MDCK) epithelial cells and antioxidant vitamin protection. Subconfluent and near-confluent MDCK cells were cultured under normoxia or hyperoxia for two days. We measured cell number and viability, mitochondria enzymatic activity, thymidine incorporation, necrosis [lactate dehydrogenase (LDH) release], and apoptosis (DNA fragmentation and morphological changes). When the cells were subconfluent, hyperoxia decreased the number of adherent cells, mitochondrial enzymatic activity, and thymidine incorporation, but neither LDH release nor apoptotic changes increased compared with normoxic controls. In normoxia, near-confluent cells had lower nonadherent cell numbers, mitochondrial enzymatic activity, and thymidine incorporation than subconfluent cells; hyperoxia further decreased the latter two parameters and increased apoptotic changes and LDH release in near-confluent cells. Vitamin E protected mitochondrial enzymatic activity, apoptotic changes, and LDH release against hyperoxic injury but did not affect changes in thymidine incorporation with hyperoxia. Vitamin C partially protected the mitochondrial enzymatic activity and thymidine incorporation in subconfluence, but not in near confluence. These results indicate that cell density is a major determinant of the effects of hyperoxic injury and the profile of antioxidant vitamin protection.

Animals↗
PubMed1997

Effects of hyperoxia on type II cell Na-K-ATPase function and expression.

Alveolar fluid is resorbed using active Na+ transport primarily through basolateral sodium-potassium-adenosinetriphosphatase (Na-K-ATPase) and apical Na+ channels that are particularly dense on the alveolar type II (ATII) epithelial cells. During lung injury with pulmonary edema, continued or accelerated Na+ and fluid resorption is critical for a favorable outcome. However, little is known of how ATII cell Na+ transport is affected during injury. These experiments examined the effects of acute lung injury on ATII cell Na-K-ATPase activity and expression using an established model of rats exposed to 100% O(2) for 60 h. Na-K-ATPase activity of ATII cells isolated immediately after exposure was assessed by ouabain-sensitive (86)Rb+ uptake in intact cells and by ouabain-sensitive P(i) production by cell membranes. In the presence of 1 mM ouabain, ouabain-sensitive Rb+ uptake was not different between normoxic and hyperoxic cells, but the apparent Na-K-ATPase maximal velocity (Vmax) of hyperoxic cell membranes was 75 +/- 8% of normoxic membranes (P < 0.05). On Western blots of ATII cell membranes, alpha1-subunit protein significantly decreased with hyperoxia (35 +/- 9% of normoxia; P < 0.05), whereas the amounts of the beta-subunit were unchanged (P > 0.05). On Northern blots of ATII cell total RNA, steady-state levels of both the alpha1- and beta1-subunit mRNA increased after hyperoxia (alpha1 = 2.5 +/- 1.3-fold; beta1 = 4.6 +/- 2.5-fold). Thus despite hyperoxic decreases in Na-K-ATPase Vmax and the amount of alpha1-protein, Rb+ uptake by Na-K-ATPase in intact cells was unchanged. The mRNA levels, protein amounts, and enzyme activity did not respond in parallel to hyperoxic injury, and the activity in intact cells correlated best with the amounts of the beta-subunit, the limiting component in de novo pump assembly in many tissues.

Animals↗
PubMed1997

Developmental changes of fetal rat lung Na-K-ATPase after maternal treatment with dexamethasone.

Late in gestation, the prenatal fetal alveolar epithelium switches from fluid secretion to resorption of salt and water via apical sodium channels and basal Na-K-ATPase. The amounts of lung sodium pump activity protein and mRNA increase in the lung just before birth. Because maternal glucocorticoids (GC) may promote maturation of the alveolar epithelium and augment fetal surfactant apoprotein levels, we hypothesized that GC increase the fetal lung Na-K-ATPase alpha- and beta-subunit gene expression in development. Timed-pregnant Sprague-Dawley rats were injected daily with intraperitoneal dexamethasone (1 mg/kg) or saline for 1, 3, or 5 days before death at fetal day (FD) 17 or 19. Maternal GC treatment altered the fetal lung wet to dry weight, decreasing it at FD17 and increasing it at FD19. Northern analysis of total lung RNA for the alpha1- and beta1-pump subunits demonstrated differential regulation of the mRNA in response to GC. At FD17, beta1-mRNA increased after 1 (FD16) or 3 days (FD14-FD16) of GC treatment, whereas alpha1-mRNA was not altered. There were accompanying increases in beta1-, but not alpha1-, protein. At FD19, GC treatment for 5 days (FD14-FD18) increased beta1- and decreased alpha1-mRNA levels, but treatment for 1 (FD18) or 3 days (FD16-FD18) had no effect. In all groups, the alpha1-Na-K-ATPase protein was predominantly on the basolateral surface of airspace epithelium by immunofluorescence. In summary, maternal dexamethasone differentially affected the fetal lung mRNA levels of the two sodium pump subunits in a complex manner, with increased beta1-mRNA levels dependent on duration of treatment and fetal age.

Animals↗
PubMed1997

Integrin mediation of alveolar epithelial cell migration on fibronectin and type I collagen.

Acute lung injury leads to type I alveolar epithelial cell (AEC) death, denudation of the alveolar basement membrane, and formation of an alveolar provisional matrix from fibronectin, fibrinogen, and type I collagen. The provisional matrix provides a scaffold for alveolar repair. To restore normal lung architecture, surviving type II AECs must reepithelialize denuded alveoli. We examined whether AECs migrate on provisional matrix proteins and whether integrins mediate this migration using a Boyden chemotaxis chamber. Cultured AECs migrated on fibronectin-coated filters by haptotaxis (defined as movement on a solid-phase substrate) more than one type I collagen-coated filters, and they did not migrate on fibrinogen-coated filters. Soluble fibronectin augmented migration on type I collagen-coated filters, but not on fibronectin-coated filters. Anti-alpha v beta 3-integrin monoclonal antibody (MAb) inhibited migration on substrate-bound fibronectin by 62-77%, whereas anti-beta 1-integrin MAb inhibited migration by 48%. Anti-alpha 2-integrin MAb almost completely inhibited migration on substrate-bound type I collagen, but not on fibronectin. The novel findings in this study are as follows: 1) AECs migrate by haptotaxis more effectively on substrate-bound fibronectin than on type I collagen; 2) alpha v beta 3- and beta 1-integrins partially mediate AEC haptotaxis on fibronectin; and 3) the alpha 2 beta 1-integrin mediates AEC migration on type I collagen. These results support the importance of type II cell migration on provisional matrix proteins during repair of lung injury.

Animals↗
PubMed1997

Hyperoxic effects on alveolar sodium resorption and lung Na-K-ATPase.

Active Na+ transport by the alveolar epithelium keeps alveoli relatively dry. Hyperoxia increases epithelial permeability, resulting in pulmonary edema. We sought to determine whether active Na+ resorption from the air spaces and Na-K-ATPase activity increased in rats exposed to > 95% O2 for 60 h. The permeability x surface area products for unidirectional resorption of alveolar [14C]sucrose (PSsucrose) and 22Na+ (PSNa+) were measured in isolated, perfused rat lungs immediately after hyperoxia and after 3 and 7 days of recovery in room air. At 60 h of hyperoxia, the mean PSsucrose and PSNa+ increased from 6.71 +/- 0.8 x 10(-5) to 12.6 +/- 1.6 x 10(-5) cm3/s (P = 0.029) and from 23.6 +/- 1.1 x 10(-5) to 31.0 +/- 1.6 x 10(-5) cm3/s (P < 0.008), respectively. However, the values in individual rats ranged widely from no change to nearly a fourfold increase. Subgroup analysis revealed that benzamil- or amiloride-sensitive (transcellular) PSNa+ was significantly reduced in the exposed lungs with normal PSsucrose but was maintained in the lungs with high PSsucrose. By day 3 of recovery, mean Na+ and sucrose fluxes returned to values similar to control. Na-K-ATPase membrane hydrolytic maximal velocity (Vmax) activity fell significantly immediately after hyperoxic exposure but recovered to normal values by day 3 of recovery. The Na-K-ATPase beta 1-subunit antigenic signal did not significantly change, whereas the alpha 1-subunit levels increased during recovery. In summary, there was a heterogeneous response of different rats to acute hyperoxia. Hyperoxia led to complex, nonparallel changes in Na+ pump antigenic protein, hydrolytic activity, and unidirectional active Na+ resorption. Active Na+ transport was differentially affected, depending on degree of injury, but permeability and transport normalized by day 3 of recovery.

Amiloride↗
PubMed1996

Hyperoxia enhances expression of gamma-glutamyl transpeptidase and increases protein S-glutathiolation in rat lung.

By participating in glutathione (GSH) synthesis, gamma-glutamyl transpeptidase (GGT) influences the GSH redox cycle, which is a major contributor in protecting against reactive oxygen metabolites. This study determined the effect of prolonged exposure of neonatal rats to > 98% oxygen on expression of GGT and on GSH metabolism. Lungs of neonatal rats chronically exposed to hyperoxia had increased expression of GGT mRNA, resulting in significantly higher GGT protein levels and enzyme activity than in lungs of animals raised in room air. Hyperoxia also upregulated glucose-6-phosphate dehydrogenase, but Na-K-ATPase activity was not changed. GGT mRNA, protein level, and enzyme activity returned to control levels after recovery in room air for 3 days. Levels of GSH, glutathione disulfide, and protein-bound GSH (S-glutathiolated protein) rose with hyperoxia and fell during recovery. S-glutathiolation is likely a mechanism for protection and a regulatory modification of protein sulfhydryl groups. Hyperoxia-induced upregulation of GGT and the concomitant increase in protein S-glutathiolation appear to be additional components fundamental in protecting the lung against oxidative injury.

Animals↗
PubMed1996

Developmental regulation of Na, K-ATPase in rat lung.

Late in gestation lung epithelium changes from net chloride and fluid secretion to sodium and fluid absorption. Fluid resorption is required for postnatal gas exchange and occurs by combined action of epithelial sodium channels and Na, K-ATPase. We hypothesized that alveolar epithelial Na, K-ATPase increases perinatally. Immunofluorescence (IF) and immunoelectron microscopy (IEM) with a monoclonal anti-alpha subunit antibody demonstrated that Na, K-ATPase was present on the basolateral surfaces of columnar epithelial cells at fetal day (FD) 17 and on type II cells throughout development. However, type I epithelial cells did not have detectable Na,K-ATPase. The steady-state levels of both the alpha 1 isoform and the beta-subunit mRNAs were maximal at FD20-neonatal day (ND) 1, with consistent increases from FD17 level. Na, K-ATPase alpha-subunit protein also increased from FD17 to FD20-22 and then decreased in the early postnatal period. The ouabain-inhibitable sodium pump activity per milligram membrane protein increased 2.6-fold from FD17 to FD22-ND1 (P < 0.05). The quantities of sodium pump mRNA, antigenic protein, and enzyme activity increase in late gestation in accord with a proposed role for Na, K-ATPase in resorption of alveolar sodium and fluid in preparation for birth.

Aging↗
PubMed1996

Integrin mediation of type II cell adherence to provisional matrix proteins.

Lung injury causes alveolar type I epithelial cell death, basement membrane denudation, and alveolar flooding with serum fibronectin and fibrinogen. For successful restoration of normal architecture, the epithelium must be regenerated from progenitor type II alveolar cells. Using adhesion assays, we examined whether type II alveolar cells adhere to the provisional matrix proteins fibronectin, fibrinogen, and fibrin, and whether integrins mediate this adherence. Rat type II cells adhered to fibronectin, vitronectin, fibrinogen, and fibrin. Synthetic RGD (arginine-glycine-aspartic acid) peptide blocked this adhesion. Flow cytometry and Western analysis indicated that type II cells expressed beta 1- and alpha v beta 3-integrins. Anti-beta 1-and anti-alpha v beta 3-integrin antibodies blocked type II cell adhesion to fibronectin and to fibronectin and fibrinogen, respectively. In summary, type II cells adhered to fibronectin, fibrinogen, and fibrin, and adhesion was partially mediated by integrins. This study provides the first evidence of type II cell adhesion to fibrin gels and vitronectin, beta 1- and alpha v beta 3-integrin mediation of type II cell adhesion, and the presence of the alpha v beta 3-integrin on type II epithelial cells.

Animals↗
PubMed1996

Terbutaline stimulates alveolar fluid resorption in hyperoxic lung injury.

Alveolar fluid resorption occurs by active epithelial sodium transport and is accelerated by terbutaline in healthy lungs. We investigated the effect of terbutaline on the rate of alveolar fluid resorption from rat lungs injured by hyperoxia. Rats exposed to > 95% O2 for 60 h, sufficient to increase wet-to-dry lung weight and cause alveolar edema, were compared with air-breathing control rats. After anesthesia, the animals breathed 100% O2 for 10 min through a tracheostomy. Ringer solution was instilled into the alveoli, and the steady-state rate of volume resorbed at 6 cmH2O pressure was measured via a pipette attached to the tracheostomy tubing. Ringer solution in some animals contained terbutaline (10(-3) M), ouabain (10(-3) M), or both. Normoxic animals resorbed 49 +/- 6 microliters.kg-1.min-1; ouabain reduced this by 39%, whereas terbutaline increased the rate by 75%. The effect of terbutaline was blocked by ouabain. Hyperoxic animals absorbed 78 +/- 9 microliters.kg-1.min-1; ouabain reduced this by 44%. Terbutaline increased the rate by a mean of 39 microliters.kg-1.min-1, similar to the absolute effect seen in the normoxic group, and this was blocked by ouabain. Terbutaline accelerates fluid resorption from both normal and injured rat lungs via its effects on active sodium transport.

Adrenergic beta-Agonists↗
PubMed1996

Cell fusion to study nuclear-cytoplasmic interactions in endothelial cell apoptosis.

Studies examining the regulation of nuclear rearrangements during apoptosis have led to conflicting results. Cytoplasmic control of nuclear events has been strongly suggested by cell-free experimental systems. In contrast, strict cytoplasmic control cannot account for the results of fibroblast-thymocyte fusion experiments in which dexamethasone induction of polykaryons led only to thymocyte nuclear apoptosis. Unresolved by these fusion studies was whether fibroblast nuclei were indifferent to heterologous cytoplasmic signals. Our objective was to resolve this discrepancy using cell fusion in a homologous system. Our strategy was to fuse endothelial cells with high levels of susceptibility to the induction of apoptosis (log phase cells arrested in G1 for 48 hours by isoleucine deprivation) with those manifesting low levels of susceptibility (serum-deprived, G0). Resultant fused and unfused cells were induced to undergo apoptosis by incubation with tumor necrosis factor-alpha and cycloheximide. Depending on the parental cell of origin, between 14 and 30% of dikaryons contained one apoptotic and one intact nucleus, indicating that strict cytoplasmic control was not occurring. In accord with this, the total frequency of nuclear apoptosis was unchanged after fusion. However, the distribution of apoptotic nuclei revealed a pronounced cytoplasmic influence, with a two- to fivefold increase in coordinate nuclear behavior. This pattern of nuclear apoptosis was consistent with a model of control in which both the state of nuclear susceptibility to apoptosis and expression of cytoplasmic pro-apoptotic regulators determined whether nuclear apoptosis would eventuate.

Animals↗
PubMed1995

gamma-Glutamyl transpeptidase is a polarized alveolar epithelial membrane protein.

In many diseases the lung is injured by oxidants. gamma-Glutamyl transpeptidase (GGT) is an ectoenzyme on the apical plasma membrane of many epithelial cells that protects against oxidants by replenishing intracellular glutathione. We sought to localize GGT within rat lungs in vivo and in cultured alveolar epithelial cells. In the adult rat lung, indirect immunofluorescence (IF) with a polyclonal antibody to triton-solubilized GGT revealed linear staining outlining the alveoli. Immunoelectron microscopy (IEM) localized the protein on the apical surface of the alveolar epithelial cells, but more densely on type I cells than type II cells, as well as on the apical surface of some ciliated bronchial cells. On Western blots of whole lung and isolated type II cell membrane proteins, the antibody predominantly recognized a broad protein band of 110-120 kDa, consistent with the uncleaved, glycosylated form of GGT. Over time in culture, isolated rat type II cells had increasing immunoreactivity on Western blots and indirect IF but decreasing enzyme activity. At 2 days in culture, confocal laser scanning microscopy demonstrated that GGT was polarized to the apical surface of nonconfluent type II cells. Thus GGT is a polarized apical membrane protein in type I and II cells, suggesting a role in the metabolic functions of these cells. The increased immunoreactive GGT of cultured type II cells is consistent with their acquisition of properties similar to type I cells, but the lack of correlation between immunoreactive protein and enzyme activity awaits explanation.

Animals↗
PubMed1994

Induction of endothelial cell apoptosis by TNF alpha: modulation by inhibitors of protein synthesis.

Our objective was to examine the induction of endothelial cell apoptosis by the proinflammatory ligand, TNF alpha. We hypothesized that TNF alpha influences endothelial cell viability by altering the balance of regulatory molecules that either induce or suppress apoptosis. Since the identity of these regulators is unknown, our approach was to examine induction of endothelial cell apoptosis by TNF alpha alone and in the context of an inhibitor of transcription or translation. TNF alpha was able to induce bovine pulmonary artery endothelial cell apoptosis in a dose-dependent fashion. Inhibition of transcription with actinomycin D or translation with cycloheximide also resulted in apoptosis, which reached a maximum value of approximately 35 to 40% of cells after 24 h. TNF alpha induction of apoptosis was either potentiated or abrogated by cycloheximide, depending on the timing of TNF alpha exposure in relation to inhibition of protein synthesis. When cycloheximide was added concomitantly with midrange concentrations of TNF alpha, there was both a dramatic acceleration and a synergistic increase in the observed apoptotic response, with all endothelial cells dying within 24 h. When cycloheximide was added as a function of time after termination of TNF alpha exposure, the synergistic induction of apoptosis was maintained at > 70% of its maximum value for 1 h, declining monotonically in a time-dependent fashion to baseline values after 6 h. In contrast, when TNF alpha was added after protein synthesis was inhibited, no additional increase in apoptosis above that observed with inhibition of protein synthesis alone was observed. Our results are consistent with the concept that endothelial cell viability depends on an interaction of inducers and suppressors of apoptosis, which are susceptible to modulation by TNF alpha.

Animals↗